A standard polymerase chain reaction (PCR)/sequencing workflow generally includes three steps requiring reagent addition: an initial PCR step, a cleanup step, and a sequencing step. The PCR step involves amplification of a template polynucleotide using amplification primers and a thermo-stable DNA polymerase enzyme. The cleanup step is commonly done by the addition of exonuclease I and alkaline phosphatase, followed by incubation, and subsequent heat-kill to inactivate the enzymes. A standard PCR/sequencing workflow is illustrated in FIG. 1.
A typical PCR reaction uses an excess of amplification primers, some which remain even upon completion of the reaction. This necessitates removal of the excess primers before proceeding to a sequencing reaction, because the excess amplification primers will interfere with a subsequent sequencing reaction. A PCR reaction furthermore contains an excess of dNTPs that can interfere with a subsequent sequencing reaction. The hydrolytic properties of exonuclease I degrade single-stranded DNA present in the PCR mixture allowing the amplification product (amplicon) to be used more efficiently in subsequent sequencing applications. The enzyme activity of alkaline phosphatase dephosphorylates dNTPs remaining from the PCR reaction. After an appropriate incubation period, the exonuclease I and alkaline phosphatase enzymes are heat inactivated before adding sequencing primer, dNTPs, and ddNTPs-dyes; otherwise the enzymes would degrade these reagents and the sequencing reaction products.
Without adequate exonuclease I and alkaline phosphatase treatment to remove excess PCR amplification primers and dNTPs, aberrant sequence ladders can be generated. For instance, as illustrated in FIG. 1, sequence ladders can be generated from both ends of a DNA sequence, causing overlapping forward and reverse sequence ladders. An excess of dNTPs can produce a weak sequencing signal and/or short sequence reads.
Problems exist in standard PCR/cycle sequencing in that the cleanup step involves an extra reagent addition step in a PCR/cycle sequencing workflow. This extra reagent addition step requires extra labor, increases the chance of introducing errors or cross contamination into the process, and can make automation difficult. A need exists for improved methods of PCR and sequencing.